In previous studies, we reconstituted microtubule-based endocytic vesicle processing in vitro using a novel fluorescence microscopy system and characterized functionally and biochemically an early endocytic vesicle population from hepatocytes in which fluorescently labeled asialoorosomucoid and its receptor colocalize, and a late population containing asialoorosomucoid but not receptor. Although both populations of vesicles move bidirectionally on microtubules following addition of ATP, only the early endocytic vesiclesundergo fission, segregating asialoorosomucoid from receptor. Little is known regarding the mechanisms by which early endocytic vesicles transition into late endocytic vesicles which ultimately fuse with lysosomes. We have prepared highly purified endocytic vesicle populations using fluorescence activated cell sorting technology and have initiated studies to characterize the proteins associated with these vesicles using proteomics-based technology. Several vesicle-specific motor molecules and Rabs have been identified and their binding partners and function will be the subject of the current proposal. We have also found that vesicles containing egressing Herpes Simplex virus intersect in part the compartments utilized during asialoglycoprotein endocytosis. In addition, we have shown that PKC is highly associated with a subpopulation of intracellular vesicles that contain the bile acid transporter, ntcp, and have shown that activity of the kinase PKC is required for their motility. We will examine whether PKC resides in a protein complex, the constituents of such a complex, whether one of these constituents is a substrate for phosphorylation by PKC, and how this might regulate motility. The Specific Aims of this proposal are: (1) To identify and characterize functionally proteins that are associated with specific populations of endocytic vesicles. (2) To define the mechanism by which early endocytic vesicles transition into late endocytic vesicles. (3) To examine the role of protein phosphorylation in regulation of vesicle motility. These proposed studies will utilize heavily the Molecular Cytology Core (Core B) and the Proteomics Core (Core C). In addition, there will continue to be substantial interactions with the other 3 Projects in this Program, providing a powerful network of investigators dedicated to elucidating mechanisms by which the hepatocyte regulates trafficking of intracellular vesicles. Ultimately, these studies may have important implications for understanding and treatment or prevention of various acquired and inheritable diseases of the liver.